CN116499839B - Wine matrix standard sample containing ochratoxin A and preparation method thereof - Google Patents
Wine matrix standard sample containing ochratoxin A and preparation method thereof Download PDFInfo
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- RWQKHEORZBHNRI-BMIGLBTASA-N ochratoxin A Chemical compound C([C@H](NC(=O)C1=CC(Cl)=C2C[C@H](OC(=O)C2=C1O)C)C(O)=O)C1=CC=CC=C1 RWQKHEORZBHNRI-BMIGLBTASA-N 0.000 title claims abstract description 32
- VYLQGYLYRQKMFU-UHFFFAOYSA-N Ochratoxin A Natural products CC1Cc2c(Cl)cc(CNC(Cc3ccccc3)C(=O)O)cc2C(=O)O1 VYLQGYLYRQKMFU-UHFFFAOYSA-N 0.000 title claims abstract description 31
- DAEYIVCTQUFNTM-UHFFFAOYSA-N ochratoxin B Natural products OC1=C2C(=O)OC(C)CC2=CC=C1C(=O)NC(C(O)=O)CC1=CC=CC=C1 DAEYIVCTQUFNTM-UHFFFAOYSA-N 0.000 title claims abstract description 31
- 238000002360 preparation method Methods 0.000 title claims abstract description 14
- 239000011159 matrix material Substances 0.000 title abstract description 17
- 238000002156 mixing Methods 0.000 claims abstract description 22
- 238000004108 freeze drying Methods 0.000 claims abstract description 21
- 241000219095 Vitis Species 0.000 claims abstract description 15
- 235000009754 Vitis X bourquina Nutrition 0.000 claims abstract description 14
- 235000012333 Vitis X labruscana Nutrition 0.000 claims abstract description 14
- 235000014787 Vitis vinifera Nutrition 0.000 claims abstract description 14
- 238000001914 filtration Methods 0.000 claims abstract description 14
- 239000000843 powder Substances 0.000 claims abstract description 14
- 239000012528 membrane Substances 0.000 claims abstract description 10
- 230000001954 sterilising effect Effects 0.000 claims abstract description 10
- 235000011389 fruit/vegetable juice Nutrition 0.000 claims abstract description 9
- 238000012258 culturing Methods 0.000 claims abstract description 8
- 238000004140 cleaning Methods 0.000 claims abstract description 4
- 238000007710 freezing Methods 0.000 claims abstract description 4
- 230000008014 freezing Effects 0.000 claims abstract description 4
- 238000000034 method Methods 0.000 claims description 40
- 239000000126 substance Substances 0.000 claims description 20
- 241000219094 Vitaceae Species 0.000 claims description 17
- 235000021021 grapes Nutrition 0.000 claims description 17
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 12
- 238000003860 storage Methods 0.000 claims description 11
- 239000000725 suspension Substances 0.000 claims description 11
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 10
- 230000001580 bacterial effect Effects 0.000 claims description 9
- 241000228212 Aspergillus Species 0.000 claims description 6
- 238000005507 spraying Methods 0.000 claims description 6
- 239000006229 carbon black Substances 0.000 claims description 5
- 238000003756 stirring Methods 0.000 claims description 5
- 230000004913 activation Effects 0.000 claims description 4
- 238000000227 grinding Methods 0.000 claims description 4
- 239000001963 growth medium Substances 0.000 claims description 4
- 229930183344 ochratoxin Natural products 0.000 claims description 4
- 229920001343 polytetrafluoroethylene Polymers 0.000 claims description 4
- 239000004810 polytetrafluoroethylene Substances 0.000 claims description 4
- 238000005406 washing Methods 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- 241000233866 Fungi Species 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 3
- 238000000855 fermentation Methods 0.000 claims description 3
- 230000004151 fermentation Effects 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 239000001965 potato dextrose agar Substances 0.000 claims description 3
- 238000007873 sieving Methods 0.000 claims description 3
- 239000008223 sterile water Substances 0.000 claims description 3
- 230000001476 alcoholic effect Effects 0.000 claims description 2
- 238000011049 filling Methods 0.000 claims description 2
- -1 polytetrafluoroethylene Polymers 0.000 claims description 2
- 238000004659 sterilization and disinfection Methods 0.000 claims description 2
- 238000001514 detection method Methods 0.000 abstract description 19
- 238000004458 analytical method Methods 0.000 abstract description 7
- 238000003908 quality control method Methods 0.000 abstract description 7
- 235000013305 food Nutrition 0.000 abstract description 6
- 238000007689 inspection Methods 0.000 abstract description 6
- 230000008901 benefit Effects 0.000 abstract description 5
- 238000004806 packaging method and process Methods 0.000 abstract description 3
- 239000000758 substrate Substances 0.000 abstract description 2
- 238000012360 testing method Methods 0.000 description 20
- 238000005259 measurement Methods 0.000 description 9
- 230000007774 longterm Effects 0.000 description 7
- 238000005070 sampling Methods 0.000 description 5
- 238000013112 stability test Methods 0.000 description 5
- 238000011077 uniformity evaluation Methods 0.000 description 5
- 238000011156 evaluation Methods 0.000 description 4
- 238000013097 stability assessment Methods 0.000 description 4
- 238000007619 statistical method Methods 0.000 description 4
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- 238000004364 calculation method Methods 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 238000009826 distribution Methods 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 231100000678 Mycotoxin Toxicity 0.000 description 2
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 230000006978 adaptation Effects 0.000 description 2
- 238000009533 lab test Methods 0.000 description 2
- 238000002844 melting Methods 0.000 description 2
- 230000008018 melting Effects 0.000 description 2
- 239000002636 mycotoxin Substances 0.000 description 2
- 238000010606 normalization Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000010998 test method Methods 0.000 description 2
- 239000003053 toxin Substances 0.000 description 2
- 231100000765 toxin Toxicity 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 1
- 244000061176 Nicotiana tabacum Species 0.000 description 1
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 1
- 241000228143 Penicillium Species 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 238000000540 analysis of variance Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 238000009924 canning Methods 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000011097 chromatography purification Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
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- 238000010586 diagram Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000556 factor analysis Methods 0.000 description 1
- 235000019674 grape juice Nutrition 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 238000011545 laboratory measurement Methods 0.000 description 1
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 231100000590 oncogenic Toxicity 0.000 description 1
- 230000002246 oncogenic effect Effects 0.000 description 1
- 239000000447 pesticide residue Substances 0.000 description 1
- 239000003495 polar organic solvent Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000012453 solvate Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000006257 total synthesis reaction Methods 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N2030/042—Standards
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Distillation Of Fermentation Liquor, Processing Of Alcohols, Vinegar And Beer (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a wine substrate standard sample containing ochratoxin A and a preparation method thereof, and belongs to the technical field of food detection. The preparation method comprises a series of steps of cleaning grape, removing stems and crushing, separating residue juice, respectively inoculating and culturing, sterilizing and freeze-drying residues and crushing, uniformly mixing grape wine with freeze-dried powder, filtering by a low-temperature membrane, secondarily uniformly mixing, packaging and the like, and the prepared matrix standard sample has the advantages of good uniformity and high stability after being subjected to strict uniformity inspection. The inspection proves that the standard product can ensure the quality of products no matter in the condition of freezing or high temperature, meets the transportation requirements of different areas, has long quality guarantee period, is an effective and reliable entity standard sample, can be used for quality control in the related detection analysis field, can also be used as a means for authenticating and checking the analysis capability of laboratories or technicians participating in detection, and has obvious economic value and market competitiveness.
Description
Technical Field
The invention relates to a wine matrix standard sample containing ochratoxin A and a preparation method thereof, belonging to the technical field of food detection.
Background
Ochratoxin a (OTA), produced mainly by some fungi of the aspergillus or penicillium genus, is a colorless crystalline compound, soluble in polar organic solvents and dilute sodium bicarbonate solutions, slightly soluble in water. The benzene solvate has a melting point of 94-96 ℃ and a crystallization melting point of 169 ℃ in dimethylbenzene, and has high chemical stability and thermal stability. Ochratoxin a is a relatively virulent mycotoxin, classified by the international agency for research on cancer as a potentially oncogenic toxin to humans (2B).
Problems with OTA contamination often occur. According to the related report, the content range of OTA in the wine in China is 0.1-5.65 mug/L. OTA in wine can adversely affect product quality by affecting yeast growth metabolism. The maximum limit value of OTA in wine is 2.0 mug/kg, which is also specified in published national standard GB2761-2017 "mycotoxin limit in food safety national Standard food". Therefore, detection of ochratoxin A in wine is necessary. The existing quality control means is to add pure ochratoxin A standard into a wine sample to be detected and then to carry out detection analysis, but the method can be influenced by the accuracy of the standard and the operation capability of experimental staff, and the accuracy of the method cannot be ensured because the OTA mostly remains in solid pomace and organisms due to adsorption and the direct labeling method cannot truly reflect the existence state of toxin in the wine. At present, studies have demonstrated that OTA degradation can also occur to some extent during the canning and storage of wine, which makes it difficult for OTA to be truly and stable in the wine matrix for long periods of time. Therefore, development of an ochratoxin A standard sample with wine as a matrix is needed to be used as a quality control means for detection.
Disclosure of Invention
The invention aims at solving the problems existing in the prior art, and provides a preparation method of a wine substrate standard sample containing ochratoxin A.
The invention also aims to provide a wine matrix standard sample containing ochratoxin A, which has the advantages of good uniformity and high stability.
In order to solve the technical problems, the invention adopts the following technical scheme:
a preparation method of a wine base standard sample containing ochratoxin A comprises the following steps:
culturing aspergillus carbon black on a potato dextrose agar culture medium plate, washing the spore after the spore is fully producedFiltering to remove mycelium to obtain spore fungus suspension; the concentration of the bacterial suspension is then adjusted to 1X 10 by using sterile water 6 Spare;
step two, taking ripe grapes, and cleaning and sterilizing the ripe grapes with alcohol;
removing stems of the grapes with the surfaces dried, crushing the grapes, squeezing the crushed grapes, and separating juice and residues;
spraying the prepared bacterial suspension on the surface of the residue uniformly, culturing in a sterile incubator at 25 ℃ for 7 days, wherein the thickness of the residue is 1cm, and the spraying amount of the bacterial suspension is 1mL/cm 2 ；
Sterilizing the cultured residues at 121 ℃ for 20 minutes, cooling to room temperature, freeze-drying, crushing, sieving with a 30-mesh sieve, and uniformly mixing the undersize substances to obtain freeze-dried powder;
step six, inoculating yeast into the juice according to 0.1g/L for fermentation at 15-20 ℃, unsealing for 8 days, adding ethanol solution to adjust the alcoholic strength to 16%, and storing at 4 ℃ to obtain fermented grape wine;
step seven, uniformly mixing the freeze-dried powder with the fermented grape wine, wherein the mixing ratio of the fermented grape wine to the freeze-dried powder is 75mL of fermented grape wine/g of freeze-dried powder, and filtering at 2-4 ℃ after uniformly mixing to obtain grape wine;
and step eight, uniformly mixing and stirring the wine for the second time, filling, and refrigerating at 2-8 ℃ for preservation.
In the first step, the culture temperature of the aspergillus carbon black is 25 ℃ and the culture time is 6 days; the filtration is carried out by sterile gauze.
In the second step, the concentration of alcohol is 75%.
In the third step, the crushing method is to crush by using a wall breaking machine; the squeezing method is to use a squeezer to squeeze.
In the fifth step, the sterilization method is to sterilize by adopting an autoclave; the freeze drying method comprises the following steps: pre-freezing at-80deg.C for 10h, freeze-drying at-40deg.C for 1h, freeze-drying at-30deg.C for 4h, freeze-drying at-20deg.C for 8h, freeze-drying at-10deg.C for 8h, freeze-drying at 0deg.C for 4h, freeze-drying at 10deg.C for 2h, and freeze-drying at 20deg.C for 1h; the crushing method comprises the following steps: in a knife grinder, grinding for 30min at the speed of 2000 r/min; the method for evenly mixing the undersize products comprises the following steps: mix with a high-efficiency mixer at 1000r/min for 20min.
In the step six, the yeast is an X16 strain, and the yeast activation method comprises the following steps: activation was carried out at 35℃for 2h.
In the seventh step, the mixing method comprises the following steps: stirring was carried out by an overhead stirrer 500r/min for 30min.
In the seventh step, the filtering method comprises the following steps: filtering with plate membrane filter of polytetrafluoroethylene PTFE membrane of 0.35-10 μm.
In step two, the grape variety includes Cabernet Sauvignon.
The wine matrix standard sample containing ochratoxin A, which is obtained by the preparation method, is characterized in that the standard sample has a valid period of 6 months under a storage environment of 4-50 ℃ and a standard value of 4.00 mug/kg.
The invention has the following beneficial effects:
1. the invention provides a preparation method of an ochratoxin A matrix standard sample in wine, which is prepared by a series of steps of washing grapes, removing stems and crushing, separating residue juice, respectively inoculating and culturing, sterilizing, freeze-drying and crushing residues, mixing wine with freeze-dried powder, filtering by a low-temperature membrane, mixing for the second time, packaging and the like according to the production process of the wine. The matrix standard sample prepared by the invention has the advantages of good uniformity and high stability after strict uniformity test. The inspection proves that the standard product can ensure the quality of products no matter in the condition of freezing or high temperature, meets the transportation requirements of different areas, has long quality guarantee period, is an effective and reliable entity standard sample, can be used for quality control in the related detection analysis field, can also be used as a means for authenticating and checking the analysis capability of laboratories or technicians participating in detection, and has obvious economic value and market competitiveness.
2. The wine matrix standard sample containing ochratoxin A is mainly applied to laboratory capability verification, internal quality control, method verification and other activities; the quality control of a laboratory is facilitated, and the accuracy of quantitative detection results is ensured; the safety control of the wine production quality is enhanced, and the pesticide residue detection level of the inspection and detection mechanism is improved.
3. Uniformity is a fundamental property of standard substances. The real sample is difficult to obtain standard sample with qualified uniformity due to the influence of various factors such as difficult preparation method, high investment cost, long investment time, undefined metabolic rule of target components and the like. The invention confirms that the juice and the residue after grape squeezing are separated and treated under the specified conditions, and the samples prepared by adding ingredients and adopting the pressurized filtration process can obtain better uniformity.
4. Stability is also an essential property of standard substances, which may lead to a change in stability of the standard sample from preparation to use involving storage and transport, and thus requires two stability assessments, one is to choose the temperature for the short-term stability assessment depending on the form of sample packaging and transport. Usually, the method is carried out under different temperature conditions, and the influence of the temperature on the characteristic value of the standard substance is examined; and secondly, under the specified storage condition, the characteristic value of the standard substance is periodically detected in a longer period, and the capability of the standard substance to be kept in a specified range is examined. The short-term stability and the long-term stability of the standard sample prepared according to the invention meet the requirements, and the effective period is 6 months under the storage environment of 4-50 ℃. The A sample of the invention in example 1 was tested in 8 laboratories and the standard value was determined to be 4.00. Mu.g/kg.
Drawings
FIG. 1 is an on-press diagram of a standard sample of wine base containing ochratoxin A according to the invention.
Detailed Description
The invention will be described in further detail with reference to the accompanying drawings and specific examples. The following examples are only illustrative of the present invention and are not intended to limit the scope of the invention.
Example 1
Standard wine base samples containing ochratoxin a were prepared at different process parameters:
group A: a preparation method of a wine matrix standard sample containing ochratoxin A comprises the following steps:
selecting and purchasing Cabernet Sauvignon type grapes collected at the same time from a Shandong tobacco stand, selecting grapes with good maturity, cleaning, sterilizing and airing the grapes with 75% alcohol, manually removing stems of the aired grapes, crushing the grapes by a wall breaking machine, squeezing the crushed grapes by a compressor, and separating juice and residues for later use. Culturing Aspergillus carbon black on culture medium plate (potato dextrose agar culture medium (Guangdong circular Kai)) at 25deg.C for 6 days, washing spores after spore production, filtering with sterile gauze to remove mycelium to obtain spore bacterial suspension, and regulating the concentration of bacterial suspension to 1×10 with sterile water 6 The sample was kept at one/mL. Uniformly spraying the prepared bacterial suspension on the surface of residues, wherein the thickness of the residues is 1cm, and the spraying amount is 1mL/cm 2 Culturing in a sterile incubator at 25℃for 7 days. Sterilizing the residue at 121deg.C for 20min, cooling to room temperature, and lyophilizing at-80deg.C for 10 hr, lyophilizing at-40deg.C for 1 hr, lyophilizing at-30deg.C for 4 hr, lyophilizing at-20deg.C for 8 hr, lyophilizing at-10deg.C for 8 hr, lyophilizing at 0deg.C for 4 hr, lyophilizing at 10deg.C for 2 hr, and lyophilizing at 20deg.C for 1 hr. Freeze-drying, transferring into a knife grinder, grinding for 30min at 2000r/min, grinding into powder, sieving with 30 mesh sieve, and mixing the undersize with high-efficiency mixer at 1000r/min for 20min to obtain freeze-dried powder. Inoculating activated yeast (Laffort Co., ltd. —X10 strain, france) into grape juice (i.e., juice) at 0.1g/L, fermenting at 20deg.C for 8 days without sealing, activating the yeast at 35deg.C for 2 hr, adding ethanol solution to adjust alcoholicity to 16%, and storing at 4deg.C. The freeze-dried powder and the fermented wine are stirred for 30min by an overhead stirrer of 500r/min, each gram of freeze-dried powder is mixed with 75mL of fermented wine, and the mixture is pressurized by a plate type membrane filter at 2 ℃ and filtered by a 10 mu m filter membrane. Mixing wine twice, stirring with an overhead stirrer at 500r/min for 20min, bottling, and refrigerating at 2deg.C.
Group B: unlike group a, group B does not adjust the alcohol content.
Group C: unlike group A, group C employed a 15 μm filter.
Group D: unlike group A, group D employed a 0.3 μm filter.
Group E: unlike group A, group E was not subjected to secondary mixing.
As shown in FIG. 1, a wine base standard sample containing ochratoxin A was obtained by the preparation method of this example.
Uniformity inspection
The uniformity test was performed on each of the above groups of samples. The detection method adopts GB 5009.96-2016 (third method immunoaffinity chromatography purification liquid chromatography-tandem mass spectrometry) for detecting ochratoxin A in food safety national standard food. The statistical method adopts a single-factor variance statistical analysis method, and three horizontal samples are subjected to uniformity test by adopting F test. The specific method is that 30 bottles are randomly extracted from samples prepared from the groups, each sample is used as an independent subsample to be detected, each subsample also needs to be subjected to three parallel result measurement, and the experimental result is taken as an average value of parallel measurement. All samples were tested under repetitive conditions in random order.
The test results are shown in tables 1 to 5.
Table 1, group A sample uniformity evaluation measurement result recording Table
Table 2, group B sample uniformity evaluation measurement result recording Table
Table 3, group C sample uniformity evaluation measurement result recording Table
Table 4, group D sample uniformity evaluation measurement result record Table
Table 5, sample uniformity evaluation measurement result record Table of group E
Stability test
And (3) detecting the stability of the samples meeting the uniformity requirement, and selecting samples of the group A for experiments. According to the JJF 1343-2022 standard substance constant value, uniformity and stability assessment, determining that a statistical method of stability test is a t-analysis test method, judging whether a sample characteristic value has a unidirectional change trend or not by adopting a characteristic value change curve along with time, and assessing the stability of a standard sample by using a linear fitting model. Stability detection involves two aspects: long-term stability (storage stability) and short-term stability (transport stability). Sampling according to sampling principle of first dense and then sparse, wherein short-term stability is set with 6 sampling time points, storage is carried out at 4 ℃ and 20 ℃ and 40 ℃, long-term stability detection is set with 6 sampling time points, and storage is carried out at 2-8 ℃.3 samples of group A were randomly drawn at each sampling time point, each sample was run in duplicate, and the average of three subsamples was taken to analyze the content of ochratoxin A in the samples of group A.
The test results are shown in tables 6 to 13.
Table 6 results of short term stability of ochratoxin A in group A samples at 4℃
Table 7 results of short term stability of ochratoxin A in group A samples at 20℃
Table 8 results of short term stability of ochratoxin A in group A samples at 40℃
Table 9, results of long term stability of ochratoxin A in group A samples
Table 10 results of short term stability of ochratoxin A in group B samples at 4℃
Table 11 results of short term stability of ochratoxin A in group B samples at 20℃
Table 12 results of short term stability of ochratoxin A in group B samples at 40℃
Table 13, results of long-term stability of ochratoxin A in group B samples
As can be seen from the uniformity test of the samples, the detection result of the samples of group D is lower than the quantitative limit, and the loss degree of ochratoxin A is too great, so that the samples have no value as standard samples. The data are subjected to single-factor analysis of variance, so that the following analysis of variance results can be obtained: at a confidence probability of 0.95, degree of freedom f 1 =29，f 2 In the case of =60, it can be derived from the F test table, F 0.05（29，60） F values in samples of groups a and B =1.65<Critical value F 0.05（29，60） The samples were uniform and required as quality control samples, indicating no significant differences between the two groups of samples. And the F values in the samples of the group C, the group D and the group E are all larger than the critical value F 0.05（29，60） Indicating that the sample was non-uniform. Stability tests of groups A and B show that the samples remain stable for a short period of time, but the samples of group B show instability in long-term stability tests. The method shows that the membrane filtration and secondary mixing have remarkable influence on the uniformity of the matrix standard sample finally prepared by the method.
As can be seen from tables 6 to 9, group A samples prepared by the method of the present invention were examined for |b in short term stability and long term stability of six months 1 All of which are smaller than t 0.95，n-2 ×s(b 1 ) The content of ochratoxin A in the matrix standard sample tends to be stable, and the storage environment of the matrix standard sample can be determined under the storage environment of 2-8 ℃, and the effective period is 6 months.
Constant value
And (3) determining the value of the sample in the group A according to the requirements of JJF 1343-2022 standard substance determination and uniformity and stability evaluation. The matrix standard sample is fixed by adopting a method of 8 labs for cooperative fixed value, and all labs participating in fixed value are qualified labs. The detection result is to detect whether the data obeys normal distribution, then check whether the data in each laboratory has suspicious value by using the Grabbs, and judge whether the data in each laboratory has the same precision by using the Keclen test, and under the condition of meeting the requirements, the data is representative and can participate in the statistical analysis of the fixed value result.
The test results are shown in Table 14.
Table 14, prepared group A Standard sample inter-laboratory measurement data
The normalization test uses Xia Piluo-wilk test, and the look-up table yields W (n, p) =0.842 (where n=10, p=0.95). Since each laboratory data pass had a W value greater than 0.842. The laboratory test data received can therefore be considered as normal distribution.
Checking whether the detection result in each laboratory group has suspicious value by using the Grabbs test method, wherein lambda is known from the Grabbs critical value table （0.05,10） =2.290. The result shows that the absolute value of the maximum residual value of the detection result is smaller than lambda （0.05,10） * S, representing the multiplication number X, indicating that no abnormal value exists among the fixed value results of each laboratory, and no suspicious value exists, so that all data are reserved and can participate in the fixed value result statistics.
And adopting a Keclen test to judge whether the constant value results of all laboratories have equal precision. The Keclen test requirement C is less than or equal to C (α,m,n) Indicating equal precision among the average values of all the groups of data, otherwise, judging the average values as outliers, and removing the group of data when calculating a constant value result. Looking up the critical table, C (0.05,8,10) = 0.2829. Table 14 shows that the C value of the Keclen test is 0.1896, which is less than the threshold, the laboratory test results are of equal accuracy, and all data should be reserved for the statistics of the constant results. The results of the 8 laboratory mean tests are shown in Table 15 below.
Table 15, 8 laboratory mean test results
According to the requirements of JJF 1343-2022 standard substance constant value, uniformity and stability evaluation, after suspicious value test and equal precision test are performed on each laboratory constant value result, whether significant difference exists or not and whether the data distribution normalization is met is also required to be checked on the average value of each laboratory constant value result. Through calculation, no significant difference exists among 8 laboratory average values and the data meet the normal inspection, and the data meet the statistical requirements, so that the standard value of the wine base standard sample containing ochratoxin A prepared in the group A is taken as the average value of the average results of all the laboratories, namely the standard value of the wine base standard sample containing ochratoxin A prepared in the group A is 4.00 mg/kg.
Uncertainty degree
According to JJF 1343-2022 "evaluation of standard substance constant value and uniformity, stability", uncertainty of standard substance constant value result is composed of 3 parts: uncertainty U caused by standard substance nonuniformity bb Uncertainty U caused by instability of standard substance sts Uncertainty U caused by standard substance constant value process char . The resultant uncertainty U (y), with a 95% level of confidence, can be determined by calculating the uncertainty for each part, with an expanded uncertainty U being twice the resultant uncertainty:
uncertainty of uniformity introduction
According to JF 1343-2022 (evaluation of standard substance's fixed value, uniformity and stability), the variance in the bottle of group A samples is smaller than the variance between the bottles of the samples) Thus, the standard deviation s of the uniformity among the bottles can be estimated bb Equivalent to the uncertainty component U caused by bottle-to-bottle non-uniformity bb . Therefore U bb = 0.00165mg/kg. But due to U bb <s 2 The estimated standard deviation between the bottles is shown to be smaller than the influence of the standard deviation between the bottles on the standard deviation between the bottles, and the uncertainty caused by the non-uniformity of the standard substance can be calculated by the following formula.
Wherein:
MS within -measurement repeatability variance in the inter-bottle uniformity assessment;
n- -number of intra-group measurements;
--MS within is a degree of freedom of (c).
The uncertainty component U caused by the uniformity finally obtained through calculation bb 0.02931. Mu.g/kg.
Uncertainty in stability introduction
According to the requirements of JF 1343-2022 standard substance constant value and uniformity and stability assessment, the sample stability variation trend of example 1 is not obvious. When the trend analysis method is adopted for stability test, the following formula can be adopted: u (U) sts =s(β 1 ) X calculates the uncertainty introduced by stability. S (. Beta.) for the monitoring of the stability of the standard substance at this time is known 1 ) X= 0.00760, x=6 months, so U sts =0.0456μg/kg。
Uncertainty of constant value introduction
The group a samples were assigned in a manner that was coordinated by multiple laboratories using a variety of methods for which accuracy was confirmed. Each laboratory only provided a series of observations. According to standard requirements, the standard deviation of the average value in such a constant value mode is in principle the constant value uncertainty U char 。
The s value is the standard deviation of the mean value of the results of the eight laboratory definite values, the p value is the total number of the laboratories participating in the definite value, and the standard uncertainty U introduced by the laboratory combined definite value is finally obtained through calculation char =0.0071μg/kg。
Therefore, the calculated total synthesis uncertainty is U （y） 0.0547mg/kg and the expansion uncertainty U was 0.1094mg/kg. The final determined characteristic value of ochratoxin a in the wine base standard samples of group a was 4.00± 0.1094 μg/kg (k=2).
Example 2
The difference between this embodiment and the group a of embodiment 1 is only that: the fermentation temperature of the yeast is 15 ℃; filtering with plate membrane filter at 4deg.C; the size of the filter membrane was 0.35. Mu.m.
The matrix standard sample obtained by the embodiment has the advantages of good uniformity and high stability after strict uniformity test.
It should be appreciated that in the above description of exemplary embodiments of the invention, various features of the invention are sometimes grouped together in a single embodiment, figure, or description thereof for the purpose of streamlining the disclosure and aiding in the understanding of one or more of the various inventive aspects. However, the disclosed method should not be construed as reflecting the intention that: i.e., the claimed invention requires more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive aspects lie in less than all features of a single foregoing disclosed embodiment. Thus, the claims following the detailed description are hereby expressly incorporated into this detailed description, with each claim standing on its own as a separate embodiment of this invention.
While the invention has been described with respect to a limited number of embodiments, those skilled in the art, having benefit of the above description, will appreciate that other embodiments are contemplated within the scope of the invention as described herein. Furthermore, it should be noted that the language used in the specification has been principally selected for readability and instructional purposes, and may not have been selected to delineate or circumscribe the inventive subject matter. Accordingly, many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the appended claims. The disclosure of the present invention is intended to be illustrative, but not limiting, of the scope of the invention, which is defined by the appended claims.
The foregoing is only a preferred embodiment of the invention, it being noted that: it will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the principles of the present invention, and such modifications and adaptations are intended to be comprehended within the scope of the invention.
Claims (9)
1. A method for preparing a wine base standard sample containing ochratoxin a, comprising the steps of:
culturing aspergillus carbon black on a potato dextrose agar culture medium plate, washing spores after the spores are fully produced, and filtering to remove hypha to obtain spore fungus suspension; the concentration of the bacterial suspension is then adjusted to 1X 10 by using sterile water 6 Spare;
step two, taking ripe grapes, and cleaning and sterilizing the ripe grapes with alcohol;
removing stems of the grapes with the surfaces dried, crushing the grapes, squeezing the crushed grapes, and separating juice and residues;
spraying the prepared bacterial suspension on the surface of the residue uniformly, culturing in a sterile incubator at 25 ℃ for 7 days, wherein the thickness of the residue is 1cm, and the spraying amount of the bacterial suspension is 1mL/cm 2 ；
Sterilizing the cultured residues at 121 ℃ for 20 minutes, cooling to room temperature, freeze-drying, crushing, sieving with a 30-mesh sieve, and uniformly mixing the undersize substances to obtain freeze-dried powder;
step six, inoculating yeast into the juice according to 0.1g/L for fermentation at 15-20 ℃, unsealing for 8 days, adding ethanol solution to adjust the alcoholic strength to 16%, and storing at 4 ℃ to obtain fermented grape wine;
step seven, uniformly mixing the freeze-dried powder with the fermented grape wine, wherein the mixing ratio of the fermented grape wine to the freeze-dried powder is 75mL of fermented grape wine/g of freeze-dried powder, and filtering at 2-4 ℃ after uniformly mixing to obtain grape wine;
step eight, carrying out secondary uniform mixing and stirring on the wine, filling, and then refrigerating and preserving at 2-8 ℃;
in the seventh step, the filtering method comprises the following steps: filtering with plate membrane filter of polytetrafluoroethylene PTFE membrane of 0.35-10 μm.
2. The method according to claim 1, wherein in the first step, the culture temperature of Aspergillus carbon black is 25℃and the culture time is 6 days; the filtration is carried out by sterile gauze.
3. The method according to claim 1, wherein in the second step, the concentration of alcohol is 75%.
4. The method according to claim 1, wherein in the third step, the crushing method is crushing by using a wall breaking machine; the squeezing method is to use a squeezer to squeeze.
5. The method according to claim 1, wherein in the fifth step, the sterilization is performed by using an autoclave; the freeze drying method comprises the following steps: pre-freezing at-80deg.C for 10h, freeze-drying at-40deg.C for 1h, freeze-drying at-30deg.C for 4h, freeze-drying at-20deg.C for 8h, freeze-drying at-10deg.C for 8h, freeze-drying at 0deg.C for 4h, freeze-drying at 10deg.C for 2h, and freeze-drying at 20deg.C for 1h; the crushing method comprises the following steps: in a knife grinder, grinding for 30min at the speed of 2000 r/min; the method for evenly mixing the undersize products comprises the following steps: mix with a high-efficiency mixer at 1000r/min for 20min.
6. The method according to claim 1, wherein in the sixth step, the yeast is an X16 strain, and the yeast activation method comprises: activation was carried out at 35℃for 2h.
7. The method according to claim 1, wherein in the seventh step, the method of mixing comprises: stirring was carried out by an overhead stirrer 500r/min for 30min.
8. The method of claim 1, wherein in step two, the grape species comprises Cabernet Sauvignon.
9. The wine base standard sample containing ochratoxin A obtained by the preparation method according to any one of claims 1-8, wherein the standard sample has a shelf life of 6 months in a storage environment of 4-50 ℃ and a standard value of 4.00 μg/kg.
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